Malignant melanoma is a devastating tumor for which new therapeutic options are needed. Despite therapeutic successes with BRAF and MEK inhibitors, resistance to these compounds is a major hurdle. Similarly, immune blockade therapies are effective, but only in a subset of populations; therefore, ways to enhance the success of immune surveillance therapies is a significant unmet clinical need. Inhibition of heat shock proteins has been an attractive avenue for melanoma, in particular because the inhibition of heat shock proteins can target cancer through multiple independent pathways, regardless of the driver oncogene(s). HSP70 inhibitors represent a new and exciting class of compounds that have shown significant pre-clinical promise for melanoma. Non-transformed melanocytes express little to no HSP70, while tumor cells express up to 100-fold higher levels, thereby providing a unique therapeutic window for the use of these inhibitors. This renewal application seeks to extend the analysis by the Murphy laboratory at The Wistar Institute with the George laboratory at the Perelman School of Medicine at the University of Pennsylvania on a novel series of HSP70 inhibitors that were discovered jointly by these laboratories. In the sixteen manuscripts published in the past funding period we showed that our series of HSP70 inhibitors target cancer cells by inhibiting the lysosome and proteasome, and that they induce cell death by causing misfolded client proteins to accumulate in insoluble aggregates, leading to proteostatic toxicity. We used proteomics to identify a dozen new HSP70 client proteins, including three with high relevance to melanoma: FAK, STAT3 and mutant BRAF. We also showed that HSP70 inhibitors also cause insolubility, aggregation and inactivation of HSP90 client proteins, due to the fact that HSP70 is an obligate co-chaperone for HSP90. We published the first crystal structure of an HSP70 inhibitor bound to substrate-binding domain of the bacterial orthologue of HSP70, DnaK. Finally, we uncovered a completely novel mechanism of action of our inhibitors: they bind to a novel allosteric pocket in HSP70, and inhibit the conformational cycles of this protein between open (ATP bound) and closed (substrate-bound) conformations, thereby inhibiting HSP70 function. In this revised renewal, the Murphy and George laboratories are joined by an expert in melanoma (Weeraratna) to assess the efficacy of HSP70 inhibition on melanoma growth, metastasis and cooperation with other melanoma therapies (BRAF inhibitors and immune checkpoint inhibitors). Support is requested for years 6-10 from this productive and collaborative team, on our novel series of HSP70 inhibitors.